The majority of cell culture studies have been performed on 2-dimensional (2D) surfaces such as micro-well plates, tissue culture flasks, and Petri dishes because of the ease, convenience, and high cell viability of 2D culture. Although these conventional 2D cell culture systems have tremendously improved our understanding of basic cell biology, they have proved to be insufficient and unsuitable for new challenges in cell biology as well as for pharmaceutical assays. Indeed, 2D culture systems fall short of reproducing the complex and dynamic environments of the in vivo situation, which are known to affect cell morphology, growth rates, contact geometries, transport properties, and numerous other cellular functions.
Three-dimensional (3D) cell culture matrices, also called scaffolds, have been introduced to overcome 2D culture limitations. These matrices are porous substrates that can support cell growth, organization, and differentiation on or within their structure. It has been demonstrated that, in comparison to conventional cultures, cells in 3D cultures more closely resemble the in vivo situation with regard to cell shape and cellular environment. Architectural and material diversity is much greater on 3D matrices than on 2D substrates. A variety of fabrication processes and biomaterials have been developed or adapted to produce cellular supports with different physical appearance, porosity, permeability, mechanical characteristics, and nano-scale surface morphology to match the diversity of in vivo environments.
A lot of efforts have focused on exploring the use of natural substances related to the extracellular matrix as biomaterials for scaffolds. Indeed, the behavior of normal and tumor cells is known to be directly conditioned by the composition of the extracellular matrix (ECM) of which hyaluronan and collagens are the principal constituents. Hyaluronan, a glycosaminoglycan composed of a repeating disaccharide of glucuronic acid and N-acetylglycosamine (β1,4-GlcUA-β1,3-GlcNAc)n, contributes significantly to cell proliferation and migration, and participates in a number of cell surface receptor interactions. It is generally accepted that hyaluronan is also implicated in tumor progression (Stern, Pathol. Biol., 2005, 53: 372-382). High contents of intracellular hyaluronan and its accumulation in the extracellular matrix create a microenvironment favorable for migration, proliferation and invasiveness of malignant cells (Delpech et al., J. Intern. Med., 1997, 242: 41-48; Toole, J. Biol. Chem., 2002, 277: 4593-4596). Thus, the invasive capability of malignant cells depends on interactions with the extracellular matrix and is promoted by hyaluronan production, as shown, for example, in the case of colon carcinoma (Kim et al., Cancer Res., 2004, 64: 4569-4576), breast adenocarcinoma (Auviven et al., Am. J. Pathol., 2000, 156: 529-536) and gastric cancers (Vizoso et al., Eur. J. Surg. Oncol., 2004, 30: 318-324). The activity of cancer cells is controlled by transductional mechanisms involving hyaluronan membrane receptors such as RHAMM or CD44. In particular, CD44, which is a ubiquitous cell surface adhesion molecule and the main receptor for hyaluronan, is implicated in cell-to-cell and cell-ECM interactions and migration of cancer cell (Assman et al., J. Pathol., 2001, 195: 191-196; Knudson et al., Matrix Biol., 2002, 21: 15-23; Ponta et al., Nat. Rev. Mol. Cell Biol., 2003, 4: 33-45). These observations have led to the use of hyaluronan as biomaterial for 3D matrices.
In particular, a reticulated hyaluronan hydrogel suitable for 3D cell culture has been developed in the laboratory of the present inventors and used to examine cancer cell invasion in 3D and evaluate cancer cell sensitivity to anticancer drugs (David et al., Matrix Biology, 2004, 23: 183-193; David et al., Cell Prolif., 2008, 41: 348-364; David et al., Acta Biomaterialia, 2008, 4: 256-263; Coquerel et al., Glia, 2009, 57: 1716-1726). However, there is always a need in the art for 3D matrices with improved properties.